ObjectivesFibrosis is a complex pathophysiological process involving interplay between multiple cell types. Experimental modelling of fibrosis is essential for the understanding of its pathogenesis and for testing of putative antifibrotic drugs. However, most current models employ either phylogenetically distant species or rely on human cells cultured in an artificial environment. Here we evaluated the potential of vascularised in vitro human skin equivalents as a novel model of skin fibrosis and a platform for the evaluation of antifibrotic drugs.MethodsSkin equivalents were assembled on a three-dimensional extracellular matrix by sequential seeding of endothelial cells, fibroblasts and keratinocytes. Fibrotic transformation on exposure to transforming growth factor-β (TGFβ) and response to treatment with nintedanib as an established antifibrotic agent were evaluated by quantitative polymerase chain reaction (qPCR), capillary Western immunoassay, immunostaining and histology.ResultsSkin equivalents perfused at a physiological pressure formed a mature, polarised epidermis, a stratified dermis and a functional vessel system. Exposure of these models to TGFβ recapitulated key features of SSc skin with activation of TGFβ pathways, fibroblast to myofibroblast transition, increased release of collagen and excessive deposition of extracellular matrix. Treatment with the antifibrotic agent nintedanib ameliorated this fibrotic transformation.ConclusionOur data provide evidence that vascularised skin equivalents can replicate key features of fibrotic skin and may serve as a platform for evaluation of antifibrotic drugs in a pathophysiologically relevant human setting.
Highly invasive animal based test procedures for risk assessment such as the Draize eye test are under increasing criticism due to poor transferability for the human organism and animal-welfare concerns. However, besides all efforts, the Draize eye test is still not completely replaced by alternative animal-free methods. To develop an in vitro test to identify all categories of eye irritation, we combined organotypic cornea models based on primary human cells with an electrical readout system that measures the impedance of the test models. First, we showed that employing a primary human cornea epithelial cell based model is advantageous in native marker expression to the primary human epidermal keratinocytes derived models. Secondly, by employing a non-destructive measuring system based on impedance spectroscopy, we could increase the sensitivity of the test system. Thereby, all globally harmonized systems categories of eye irritation could be identified by repeated measurements over a period of 7 days. Based on a novel prediction model we achieved an accuracy of 78% with a reproducibility of 88.9% to determine all three categories of eye irritation in one single test. This could pave the way according to the 3R principle to replace the Draize eye test.
Background:The complex pathophysiological processes that result in fibrotic tissue remodeling in systemic sclerosis involve interplay between multiple cell types (1). Experimental models of fibrosis are essential to provide a conceptual understanding of the pathogenesis of these diseases and to test antifibrotic drugs. Current models of fibrosis have important limitations: thein vivomodels rely on species that are phylogenetically distant, whereas thein vitromodels are oversimplified cultures of a single cell type in an artificial two-dimensional environment of excessive stiffness, which imposes an unphysiological cell polarization (2).Objectives:Here we evaluated the potential use of vascularized, three-dimensionalin vitrohuman skin equivalents as a novel model of skin fibrosis and a platform for the evaluation of antifibrotic drugs.Methods:Skin equivalents were generated by seeding human endothelial cells, fibroblasts and keratinocytes on a decellularized porcine extracellular matrix with perfusable vascular structure. The skin models were cultured for one month in a system that ensured perfusion of the vascular network at physiological pressure. Fibrotic transformation induced by TGFβ and response to nintedanib as an established antifibrotic drug was evaluated by capillary Western immunoassays, qPCR, histology and immunostaining.Results:The vascularized human skin equivalents formed the major skin structures relevant for the pathogenesis of fibrosis: a polarized, fully matured epidermis, a stratified dermis and a perfused vessel system with small capillaries. Exposure to TGFβ led to the fibrotic transformation of the skin equivalents, with activated TGFβ downstream pathways, increased fibroblast-to-myofibroblast transition and excessive deposition of extracellular matrix. Treatment of models exposed to TGFβ with nintedanib (a drug with proven antifibrotic effects) ameliorated the fibrotic transformation of skin equivalents with reduced TGFβ signaling, fibroblast-to-myofibroblast transition and decreased extracellular matrix deposition.Conclusion:Here we describe a novelin vitromodel of skin fibrosis. Our data show that vascularized skin equivalents can reproduce all skin layers affected by fibrosis, that, upon exposure to TGFβ, these models recapitulate key features of fibrotic skin and that these skin models can be used as a platform for evaluation of antifibrotic drugs in a setting with high relevance for human disease.References:[1]Distler JHW, Gyorfi AH, Ramanujam M, Whitfield ML, Konigshoff M, Lafyatis R. Shared and distinct mechanisms of fibrosis. Nature reviews Rheumatology. 2019;15(12):705-30.[2]Garrett SM, Baker Frost D, Feghali-Bostwick C. The mighty fibroblast and its utility in scleroderma research. Journal of scleroderma and related disorders. 2017;2(2):69-134.Disclosure of Interests:Alexandru-Emil Matei: None declared, Chih-Wei Chen: None declared, Lisa Kiesewetter: None declared, Andrea-Hermina Györfi: None declared, Yi-Nan Li: None declared, Thuong Trinh-Minh: None declared, Toin van Kuppevelt: None declared, Jan Hansmann: None declared, Astrid Juengel: None declared, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB, Florian Groeber-Becker: None declared, Jörg Distler Grant/research support from: Boehringer Ingelheim, Consultant of: Boehringer Ingelheim, Paid instructor for: Boehringer Ingelheim, Speakers bureau: Boehringer Ingelheim
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